USPTO Patents Application 09741664

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Whatls Claimed Is:

1 . A stable composition comprising a mixture of reagents at working
concentrations, wherein said reagents are at least one thermostable enzyme and
at least one buffer salt.

2. A stable composition for nucleic acid amplification comprising a
mixture of reagents, wherein said reagents are at least one thermostable DNA
polymerase, at least one buffer salt and at least one deoxynucleoside triphosphate.

3. A stable composition for nucleic acid sequencing comprising a
mixture of reagents, wherein said reagents are at least one thermostable DNA
polymerase, at least one deoj^ucleoside triphosphate, at least one
dideoxynucleoside triphosphate and at least one buffer salt.

4. The stable composition of claim 2 or claim 3 , wherein said reagents
are present at working concentrations.

5 . The composition of claim 2 or claim 3, wherem said thermostable
DNA polymerase is selected fi-om the group of thermostable DNA polymerases
consisting of a Taq DNA polymerase, a Tne DNA polymerase, a Tma DNA
polymerase, and mutants thereof

6. The composition of claim 2 or claim 3, wherein said thermostable
DNA polymerase is selected from the group of thermostable DNA polymerases
consisting of a Pfu DNA polymerase, a Pwo DNA polymerase, VENTt** DNA
polymerase, DEEPVENT™ DNA polymerase, and mutants thereof

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7. The composition of claim 5, wherein said mixture flirther comprises
DEEPVENT™ DNA polymerase or VENT™ dNA polymerase.

8. The composition of claim 5, wherein the concentration of Taq
DNA polymerase or mutant thereof is about 0. 1 to 200 units per milliliter.

9. The composition of claim 8, wherein the concentration is about 20
units per milliliter.

10. The composition of claim 5, wherein the concentration of Tm
DNA polymerase or mutant thereof is about 0. 1 to 200 units per milliliter.

1 1. The composition of claim 10, wherein the concentration is about
20 units per milliliter.

12. The composition of claim 5, wherein the concentration- of Tma
DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter.

13. The composition of claim 12, wherein the concentration is about
20 units per milliliter.

14. The composition of claim 6, wherein the concentration of VENT™
DNA polymerase or mutant thereof is about 0. 1 to 200 units per milliliter.

15. The composition of claim 14, wherein the concentration is about
20 units per milliliter.

16. The composition of claim 6, wherein the concentration of
DEEPVENTTM DNA polymerase or mutant thereof is about 0. 1 to 200 units per
milliliter.

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17. The composition of claim 16 wherein the concentration is about 20
units per milliliter.

18. The composition of claim 6, wherein the concentration ofP/uUNA
5 polymerase or mutant thereof is about 0.1 to 200 units per milliliter.

19. The composition of claim 1 8 wherein the concentration is about 20
units per milliliter.

10 20. The composition of claim 6, wherein the concentration of Pwo

DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter.

2 1 . The composition of claim 20 wherein the concentration is about 20
units per milliliter.

15

22. The composition of claim 7, wherein the concentration of
DEEPVENT™ DNA polymerase or VENT DNA polymerase is about 0.002 to
200 units per milliliter.

20 23. The composition of claim 22, wherein the concentration is about

0.40 units per milliliter.

24. The composition of claim 2 or claim 3, wherein said DNA
polymerase retains at least 90% of the enzymatic activity for at least four weeks

25 when stored at about 20 ° C to 25 ° C.

25. The composition of claim 5, wherein said DNA polymerase retains
at least 90% of the enzymatic activity for at least one year when stored at about
4°C.

30

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26. The composition of claim 2 or claim 3, fUrther comprising a
magnesium salt.

27. The composition of claim 2 or claim 3, further comprising at least
5 one nonionic detergent.

28. The composition of claim 2 or claim 3, wherein the concentration
of said deoxynucleoside triphosphate is about 200 to about 300 micromolar.

10 29. The composition of claim 3, wherein the concentration of said

dideoxynucleoside triphosphate is about 0.08 to about 5 micromolar.

30. A nucleic acid amplification kit comprising one or more containers,
wherein a first container contains a stable composition comprising a mixture of

15 reagents, wherein said reagents are at least one thermostable DNA polymerase,

at least one buffer salt, and at least one deoxynucleoside triphosphate.

31. A nucleic acid sequencing kit comprising one or more containers,
wherein a first container contains a stable composition comprising a xnixture of

20 reagents, wherein said reagents are at least one thermostable DNA polymerase,

at least one buffer salt, at least one deoxynucleoside triphosphate and at least one
dideoxynucleoside triphosphate.

32. The kit of claim 30 or 31, wherein said reagents are present at
25 working concentrations.

33. A method of amplifying a nucleic acid molecule comprising
contacting said nucleic acid molecule with the composition of claim 2.

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34. A method of amplifying a nucleic acid molecule comprising
contacting said nucleic acid molecule with a composition selected from the group
consisting of :

a composition comprising a thennostable 3' exo+ DNA polymerase and a
thermostable 3' exo- DNA polymerase wherein the concentrations of said 3' exo+
DNA polymerase and of said 3' exo- DNA polymerase are equal, and

a composition comprising a thermostable 3' exo+ DNA polymerase and a
thermostable 3' exo- DNA polymerase wherein the concentration of said 3' exo+
DNA polymerase is higher than the concentration of said 3' exo- DNA
polymerase.

35. A method of sequencing a nucleic acid molecule comprising
contacting said nucleic acid molecule with the composition of claim 3.

36. A method of sequencing a nucleic acid molecule comprising
contacting said nucleic acid molecule with a composition selected from the group
consisting of :

a composition comprising a thermostable 3' exo+ DNA polymerase and a
thermostable 3' exo- DNA polymerase wherein the concentrations of said 3' exo+
DNA polymerase and of said 3' exo- DNA polymerase are equal, and

a composition comprising a thermostable 3' exo+ DNA polymerase and a
thermostable 3' exo- DNA polymerase wherein the concentration of said 3' exo+
DNA polymerase is higher than the concentration of said 3' exo- DNA
polymerase.

37. The method of any one of claims 33-36, wherein said nucleic acid
molecule is larger than about 4 kilobases in size.

38. The method of claim 37, wherein said nucleic acid molecule is
larger than about 7 kilobases in size.

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39. The method of claim 38, wherein said nucleic acid molecule is
larger than about 8 kilobases in size.

40. A nucleic acid molecule amplified by the method of claim 33 or
claim 34.

41. The nucleic acid molecule of claim 40, wherein said nucleic acid
molecule is larger than about 4 kilobases in size.

42. The nucleic acid molecule of claim 41, wherein said nucleic acid
molecule is larger than about 7 kilobases in size.

43. The nucleic acid molecule of claim 41, wherein said nucleic acid
molecule is larger than about 8 kilobases in size.

44. The composition of claim 1, further comprising at least one
antibody that specifically binds to said thermostable enzyme.

45 . The composition of claim 2 or claim 3 , further comprising at least
one antibody that specifically binds to said thermostable enzyme.

46. The kit of claim 3 0 or claim 3 1 , wherein said mixture of reagents
further comprises at least one antibody that specifically binds to said thermostable
DNA polymerase.

47. The kit of claim 30 or claim 31, further comprising one or more
additional containers containing at least one antibody that specifically binds to said
thermostable DNA polymerase.